Anouk Donners

70 Chapter 4 We have recently developed and published a novel method to determine the human FVIII plasma concentration with liquid chromatography-tandem mass spectrometry (LC-MS/MS) [13]. The LC-MS/MS technique enables quantification of the FVIII molecule with a high sensitivity and specificity. Although this method is further upstream than activity measurements and has its shortcomings as well, the new method might also have some advantages. For example, sampling could be done by patients themselves at home using the dried blood spot technique. The primary objective of this proof of principle study is therefore to investigate the correlation between FVIII activity measured with OSA compared to FVIII plasma concentration measured with LC-MS/MS in patients with haemophilia A, and to identify determinants for differences between the two methods. MATERIALS AND METHODS Setting and participants This cross-sectional study was conducted at the University Medical Center Utrecht, and specifically at the laboratories of the Department of Clinical Chemistry and Haematology and the Department of Clinical Pharmacy. All haemophilia A patients or female carriers receiving standard-of-care treatment were eligible for inclusion. Their remnant material was stored in accordance with the local opt-out procedure. Within each of the clinically used FVIII activity categories (<1 IU/dL, 1–5 IU/dL, >5–40 IU/dL, >40–150 IU/dL, and >150– 600 IU/dL) 15–20 samples were randomly selected in the period of August 2017 to March 2018. The FVIII plasma concentration was measured with LC-MS/MS and compared to the FVIII activity measured with OSA. Per patient, a maximum of one sample was included per category. The local institution’s ethics committee approved the procedure and provided a waiver for patients’ consent. This study was conducted in accordance with the current revision of the Declaration of Helsinki as revised in 2013. FVIII activity with OSA After blood had been drawn for usual care, the FVIII activity was measured at the ISO15189-certified Laboratory of Clinical Chemistry and Haematology and reported as a percentage of FVIII activity compared to reference plasma. The FVIII activity was measured on a STA-Rack evolution coagulation analyser using STA CK Prest aPTT reagent (Diagnostica Stago, Asnières-sur-Seine, France). The FVIII-deficient plasma and reference plasma were obtained from Precision Biologic (Dartmouth, NS, Canada). The assay had a within-run %CV of 3.8% at a FVIII activity of 1.3 IU/dL, a between-run %CV of 4.6% at a FVIII activity of 34 IU/dL, a between run %CV of 5.6% at a FVIII activity of 88 IU/ dL, a linearity range <1–600 IU/dL, a recovery of 100.7%, and a 1.0 IU/dL lower limit of quantification. Robust internal and external quality assessment schemes were performed on the assay, and local performance characteristics were within the pre-defined limits stated by the manufacturers.